Binder for C-reactive protein

ABSTRACT

A polypeptide dimer is provided wherein both protomers have a sequence according to SEQ ID NO: 1 and at least one phosphocholine derivative is attached to the polypeptide. The polypeptide shows a specific binding for C-reactive protein (CRP). The utilization of the polypeptide in assays for determining the concentration of CRP is described. The purification of CRP, and compositions comprising the CRP also are provided.

FIELD OF THE INVENTION

The present invention relates to the field of molecules bindingspecifically to a certain protein, in this case C-reactive protein, anduses thereof.

BACKGROUND OF THE INVENTION

The de novo design of folded polypeptides aims at improving ourunderstanding of protein structure, and also provides a platform for theengineering of new proteins with tailored functions. Designed, foldedpolypeptides that undergo pH-controlled, site-selectiveself-functionalization with ligands constitute an excellent toolbox forthe construction of various complex molecular systems, e.g. modelglycoproteins or complex receptors.

The object of international patent publication WO03/080653 was toprovide folded, ligand modified helix-loop-helix polypeptide scaffoldsthat connect the key biosensing events of recognition and reporting. Thewell characterized interaction between the enzyme human carbonicanhydrase II, HCAII, and its inhibitor 4-carboxybenzenesulfonamide wasselected for a proof of principle demonstration.

C-reactive protein is a plasma protein that circulates in the bloodstream in increased amounts during inflammation and after tissue damage.CRP-levels are measured for assessment of i.a. inflammation andincreased risk of cardiovascular disease. It is also of interest tomeasure CRP-levels during heart surgery.

SUMMARY OF THE INVENTION

The present invention relates to polypeptide scaffolds further modifiedto specifically bind to C-Reactive Protein (CRP). Such modifiedpolypeptide scaffolds are hereinafter referred to as “CRP-specificbinders”, “CRP-binders” or simply “binders”, which terms are usedinterchangeably unless otherwise indicated.

In a first aspect, the invention relates to CRP-specific binderscomprising a polypeptide having a sequence according to SEQ ID NO: 1,wherein at least one phosphocholine derivative is attached to saidpolypeptide and to a polypeptide scaffold consisting of a four-helixbundle formed of dimers of polypeptides having the sequence according toSEQ ID NO: 1, which binders are modified by incorporation of a bindingmoiety comprising a phosphocholine derivative and optionally a reportergroup that can give a detectable signal. The two protomers of the dimermay be covalently attached to each other through, for example, adisulfide bond, but may also be non-covalently bound to each other. Theindividual protomers may also be used as binders.

The invention also relates to a method for producing the polypeptideaccording to the first embodiment, said method comprising the steps

-   -   synthesizing a polypeptide having the sequence according to SEQ        ID NO: 1;    -   bringing a phosphocholine derivative in contact with an        unblocked lysine of said polypeptide under conditions suitable        for the attachment of the phosphocholine derivative to the        lysine;    -   optionally bringing a reporter group in contact with an        unblocked lysine of said polypeptide under conditions suitable        for the attachment of the reporter group to the lysine.

In a further aspect the invention relates to the use of bindersaccording to the first aspect in therapeutic and diagnosticapplications, such as the incorporation of such binders inpharmaceutical or diagnostic compositions, assays for determining theconcentration of CRP in a sample, e.g. from a patient undergoing heartsurgery or suspected of having an inflammation, and in biotechnologicalapplications, such as protein purification or binding assays.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an overview of a library of peptide scaffolds (SEQ ID NOs.3-18) used in the present invention.

FIG. 2 shows the affinity of a CRP-binder of the present invention toCRP.

FIG. 3 is a schematic view of a CRP detection assay using the CRP-binderof the present invention.

FIG. 4 shows the activity of native pyruvate kinase, pyruvate kinasebound to a linker, and pyruvate kinase bound to a CRP-binder,respectively.

FIG. 5 is a comparison of signals generated by kinase-bound CRP-binderof the present invention and kinase-bound CRP-specific antibody,respectively.

FIG. 6 is a comparison of signals generated by a kinase-bound CRP-binderof the present invention used for detection of CRP from human, horse anddog.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly shown by the present inventors that the bindingaffinity between CRP and a polypeptide scaffold may be dramaticallyenhanced by attaching a phosphocholine derivative in certain positions.Phosphocholine alone has a binding affinity of ˜1 μM and the scaffoldpolypeptides have a binding affinity of ˜100-1000 μM, while thepolypeptides modified with a phosphocholine derivative have a bindingaffinity for CRP in the nanomolar range.

The unmodified scaffold binds to the surface of CRP and thephosphocholine derivative should be introduced in a position within, orin close proximity of, the binding site on the polypeptide scaffold. Inorder to reach its binding site on CRP, the phosphocholine derivativemay comprise a spacer of 1-12 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or 12 carbon atoms.

A further surprising advantage of the binders of the present inventionis that they bind with high affinity to CRP from different mammals, incontrast to CRP-specific antibodies that usually bind with high affinityto CRP from one mammalian species but with lower affinity to CRP fromother species.

The polypeptide scaffold may also be varied with regard to charge inorder to optimize binding affinity to CRP. This is preferably done byexchanging a charged amino acid residue for an uncharged residue, or aresidue with different charge, in the polypeptide, or vice versa. Forexample, a Val or Ala (uncharged) can be exchanged for a Glu (charge −1)or an Arg (charge +1).

According to one embodiment of the present invention, the polypeptidescaffold has at least two lysine residues to which a CRP-binding moietyand a reporter group, respectively, may be attached.

The basic polypeptide scaffold protomer has the sequence according toSEQ ID NO: 1. This protomer can be produced by any method known to theperson skilled in the art, such as by recombinant technology orconventional automated solid phase peptide synthesis. One or morepositions should carry a lysine. These positions are preferably selectedfrom positions 8, 10, 15, 17, 22, 25, 34 and 37. The CRP-binding moietyand the optional reporter group are then attached to the lysinesaccording to the method discussed below.

The reporter group may be any group that can be attached to a lysineresidue and give a detectable signal. Preferred examples of reportergroups are fluorescent probes such as dansyl, coumarin, fluorescein,rhodamine and Oregon Green derivatives. The reporter group may also bean enzyme such as phosphoenolpyruvate kinase or a detectable particlesuch as a gold nanoparticle. The reporter group is attached to thelysine residue according to the supplier's instructions or by otherconventional methods. Preferably, the reporter group is attached to theprotomer while the newly synthesized protomer is still on the resin.

The CRP-binding moiety is a phosphocholine derivative comprising aphosphocholine moiety and a spacer. The length of the spacer depends onwhere the ligand is attached to the scaffold and where the ligand bindsto CRP. The spacer is usually an aliphatic chain of 1-12 carbon atomsoptionally substituted with hydrophilic groups to enhance solubility.

The phosphocholine derivative is attached to the protomer by bringingthe protomer into contact with an active ester of the phosphocholinederivative. The active ester has the general formulaOP(OH)₂O(CH₂)₂N⁺(CH₃)₂(CH₂)_(n)COOR¹  (I)wherein 1≦n≦12 and R¹ is a leaving group with a pK_(a) of about 6-8. Thesynthesis of active esters wherein n is 4, 6 or 11 and R¹ isp-nitro-phenyl are described in the experimental section below.

The phosphocholine derivative is incorporated into the polypeptidescaffold as disclosed in the experimental section.

The CRP-binder of the present invention may also be bound to a solidsupport, optionally through a linker. A number of solid supports, suchas chips, plates, beads and membranes, and linkers are commerciallyavailable and may be combined by the skilled person depending on thecircumstances the CRP-binder of the invention is to be used under.

Binding Assays

One aspect of this invention relates to binding assays using the bindersof the present invention, and, more particularly, to a method foreliminating interference resulting from rheumatoid factors or humananti-mouse antibodies (HAMA) in biological samples.

Different types of sandwich assays are widely used. One of the mostcommon is sandwich ELISA where an antibody is used to capture theantigen and another labelled antibody is used to detect the boundantigen. Both of these antibodies are usually of mammalian origin. Insuch assays anti-mammalian IgG antibodies present in the samples, whichoften are derived from body fluids such as blood or serum, may simulatethe behaviour of the antigen by linking the detection antibody to thecapture antibody, thus causing false positive reactions. Such falsepositive reactions are observed in sandwich assays based on bothpolyclonal and monoclonal antibodies.

An increasing number of patients are given mouse monoclonal antibodiesfor diagnosis or therapy. Monoclonal antibody administration ofteninduces an antibody response in the patients¹²; human anti-murineantibodies (HAMA) are produced. Sandwich-type assays based on mousemonoclonal antibodies are known to give false positive results when HAMAis present in the serum of the patient. This will be an increasingproblem in the clinical chemistry laboratory as the number of patientstreated with monoclonal antibodies increases.

Rheumatoid factor (RF) is also a major source of interference in manyimmunoassays. RF is an autoantibody that reacts with the Fc portion ofmammalian IgG. RF can be of the IgA, IgG, or IgM classes. Between 60%and 80% of patients with Rheumatoid Arthritis (RA) have RF activity intheir serum. RF has also been found in serum from patients with otherconnective tissue diseases and in many other diseases. Although RFreacts with the Fc-part of IgG, they display heterogeneity in theirreactivity to IgG of different species. This reactivity to IgG of otherspecies may produce false reactions in assays involving antibodies. Theclassical tests for RF are agglutination tests, in which the RF reactswith two (or more if it is an IgM-RF) different mammalian IgG molecules,thus causing agglutination. The same reaction will give a false-positivereaction in a sandwich ELISA, owing to the binding of detection antibodyto capture antibody in the absence of the antigen that the assay wasdesigned to detect.

One approach to the above problem is the use of Fab fragments in theassay, because the anti-IgG antibodies are usually directed against theFc part of the IgG molecule. However, the digestion of IgG to producepure Fab fragments is time consuming and usually results in loss ofantibody titre and will not reduce the interference caused by anti-Fabantibodies. Furthermore, addition of mouse IgG or mouse serum orchromatography to remove the interfering antibodies have been used toavoid the above mentioned problem. Addition of normal mouse IgG ornormal mouse serum sometimes fails to eliminate the false positivereaction. Removal of interfering antibodies by chromatography is timeconsuming and less suitable for routine analysis.

One aspect of the present invention is therefore to change at least oneof the antibodies in the assay to a binder according to the inventionthat is not bound to by HAMA or RF.

In one embodiment of this aspect, the invention relates to a method forassaying the concentration of C-reactive protein in a sample, comprisingthe steps

-   -   bringing the sample in contact with a first polypeptide dimer        according to any of claims 1-6, said first polypeptide being        bound to a solid support,    -   bringing the sample in contact with a second polypeptide dimer        according to any of claims 3-6 having a reporter group attached        to it; and    -   detecting the presence or absence of the reporter group on said        second polypeptide dimer.

EXPERIMENTAL SECTION Synthesis of Phosphocholine Derivatives PC6, PC11and PC4

The phosphocholine derivative to be attached to the polypeptide scaffoldmay have a spacer of 1-12 carbon atoms, as explained above. These aredenoted PC, for phosphocholine, followed by a number giving the lengthof the spacer, i.e PC6 has a spacer of 6 carbon atoms.

For the synthesis of PC6 we originally chose a simple and short route asoutlined in Scheme 1. In the first step 6-dimethylamino hexanoic acidwas prepared by alkylation of the corresponding amino acid withformaldehyde under catalytic hydrogenation conditions in good yield. Theresulting tertiary amine was simply purified with a strong acidic cationexchanger.³ The carboxylic acid function was then esterified to form themethyl ester in essentially quantitative yield and no purification stepwas required. The subsequent alkylation with the bromide, which waseasily prepared from diethyl chlorophosphate and 2-bromo ethanol,⁴proceeded usually with only moderate yields. After RP HPLC purificationthe methyl ester was cleaved with Lithium hydroxide and the carboxylicacid was obtained in almost quantitative yield and purification was notnecessary. The generation of the active ester was done following aprocedure of Kim et al. utilizing p-nitrophenyl chlorofomate, a base andDMAP.⁵ The active ester was obtained after RP HPLC purification in 28%yield only. The final deprotection of the phosphate group was done undermild conditions with TMSBr without affecting the carboxylic ester.⁶ Onthe other hand, the formation of side products for some substratesduring this reaction is a known disadvantage of this procedure.⁷However, we obtained the desired phosphate in 25% yield besides themono-deprotected phosphate and the quaternary amine without phosphategroup.

For the preparation of PC11 we chose a synthetic route including somesteps on solid phase in order to avoid cumbersome purificationprocedures and solubility problems (Scheme 2). Fmoc-protected undecanoicacid was therefore coupled to Wang resin utilizing the DIC method in 66%yield.⁸ After deprotection with piperidine the dimethylation wasaccomplished with formic acid and sodium borohydride.⁹ The alkylationstep to the quaternary amine was a sluggish reaction and needed 10equivalents of the bromide and heating for 4 days.¹⁰ After cleavage fromthe resin the crude product was transformed to the active ester by thesame method that we used for PC6. The final deprotection step of thephosphate group proceeded similarly to PC6 with formation of byproducts,but 27% of the desired product were obtained.

Scheme 1

PC6-pNitrophenylester Synthesis

Phosphoric acid 2-bromo-ethyl ester diethyl ester. A solution of 2-bromoethanol (1.42 mL, 20 mmol) and dry pyridine (3.23 mL, 40 mmol) in drydichloromethane (25 mL) was cooled to 0° C. Diethyl chlorophosphate(3.36 mL, 23 mmol) was added dropwise and the reaction mixture wasstirred at room temperature for 24 h. Diethyl ether (40 mL) and 1 N HCl(40 mL) were added and the organic layer was separated and washedconsecutively with 1 N HCl and saturated NaHCO3 and dried over MgSO4.After evaporation of the solvents the residue was purified by columnchromatography (silica gel, ethyl acetate/pentane 1:1) to give theproduct (4.85 g, 18.6 mmol, 93%) as light yellowish oil. ¹H NMR (CDCl₃,400 MHz) δ=1.34 (dt, 6H, 2×CH₃, ³J(H,H)=7.2 Hz, ⁴J(H,P)=1.2 Hz); 3.52(t, 2H, CH₂Br, ³J=6 Hz); 4.13 (dq, 4H, 2×CH₂CH₃, ³J(H,H)=7.2 Hz,³J(H,P)=7.2 Hz); 4.28 (dt, 4H, 2×OCH₂CH₂Br, ³J(H,H)=6.4 Hz, ³J(H,P)=8.4Hz). ¹³C NMR (CDCl₃, 100.5 MHz) δ=16.0 (d, 2×CH₃, ³J=6.8 Hz); 29.4 (d,CH₂Br, ²J=7.6 Hz); 64.0 (d, OCH₂, ²J=5.3 Hz); 66.5 (d, OCH₂, ²J=5.3 Hz).

6-dimethylamino hexanoic acid. A solution of the amino acid (1.32 g, 10mmol) in water was hydrogenated in a Parr apparatus in the presence ofan excess of formaldehyde (4 mL) and 10% Pd/C (0.5 g) at 90 psi of H2 atroom temperature for 36 h. The catalyst was removed by filtration andwas washed twice with hot water. The combined aqueous layer was passedthrough a DOWEX 50WX2-100 ion exchanger column. After washing with waterthe column was eluted with 2% of aqueous NH4OH to give the pure product(1.39 g, 8.7 mmol, 87%) as colorless crystals.

6-dimethylamino hexanoic acid methyl ester. To an ice-cold solution of6-dimethylamino hexanoic acid (1.37 g, 8.6 mmol) in dry methanol (50 mL)was added thionylchloride (9 mL) dropwise over 30 min. The reactionmixture was warmed to room temperature and was stirred over night. Thesolvent was evaporated under reduced pressure. Repeated addition ofmethanol and subsequent evaporation gave the methyl ester in almostquantitative yield in pure form as colorless solid. ¹H NMR (DMSO de, 400MHz) δ=1.27 (m, 2H, CH₂); 1.53 (m, 2H, CH₂); 1.63 (m, 2H, CH₂); 2.31 (t,2H, ³J=7.6 Hz, CH₂); 2.67 (s, 6H, N(CH₃)₂); 2.96 (t, 2H, ³J=8 Hz, CH₂);3.57 (s, 3H, COOCH₃). ¹³C NMR (DMSO d₆, 100.5 MHz) δ=23.1; 23.8; 25.3;32.9; 41.7; 51.2; 56.0; 173.1.

[2-Diethoxy-phosphoryloxy)-ethyl]-(5-methoxycarbonyl-pentyl)-dimethylammoniumtrifluoroacetate. The methyl ester (1.73 g, 10 mmol) was dissolved indry acetonitrile (15 mL) and K₂CO₃ (1.38 g, 10 mmol) was added. Thissuspension was refluxed under nitrogen gas for 36 h. After cooling toroom temperature water was added and the pH was adjusted to 2 by addingTFA. The solution was purified by rp HPLC (Supelco Discovery C18, 21.2mm×15 cm, 5 μm, A: 5% IPA, 95% H₂O, 0.1% TFA, B: 90% IPA, 10% H₂O, 0.1%TFA, 0 to 15% B over 14 min, t_(R)=11.8 min) and lyophilized to give theTFA salt of the quaternary amine as light yellow oil (1.06 g, 3.0 mmol,30%). LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formicacid in water, B: 0.1% formic acid in acetonitrile, 10-50% B over 10min) t_(R)=6.4 min (LSD signal), m/z=354.2 for [M+H]⁺. ¹H NMR (CD₃OD,400 MHz) δ=1.41-1.50 (m, 8H, 2×OCH₂CH₃ and CH₂); 1.77 (m, 2H, CH₂); 1.90(m, 2H, CH₂); 2.44 (t, 2H, CH₂, ³J=7.2 Hz); 3.25 (s, 6H, N(CH₃)₂); 3.48(m, 2H, CH₂); 3.72 (s, 3H, COOCH₃) 3.82 (m, 2H, CH₂); 4.26 (dq, 4H,2×CH₂CH₃, ³J(H,H)=6.8 Hz, ³J(H,P)=8.0 Hz); 4.58 (brs, 2H, CH₂). ¹³C NMR(CD₃OD, 100.5 MHz) δ=16.4 (d, 2×OCH₂CH₃, ³J=6.1 Hz); 23.3 (CH₂); 25.3(CH₂); 26.7 (CH₂); 34.4 (CH₂); 52.0 (CH₃); 52.2 (CH₃); 62.1 (d, CH₂,²J=5.3 Hz); 64.8 (CH₂); 66.1 (d, CH₂, ²J=6.1 Hz); 66.7 (CH₂); 175.5(CO).

(5-carboxy-pentyl)-[2-(diethoxy-phosphoryloxy)-ethyl]-dimethylammoniumtrifluoroacetate. The TFA salt of the quaternary amine (200 mg, 0.56mmol) was dissolved in a mixture of t-butanol/water (2:1, 9 mL) andLiOH.H₂O (47 mg, 1.12 mmol) was added. The reaction mixture was stirredfor 3 h at room temperature and the organic solvent was removed underreduced pressure. The pH of the aqueous solution was adjusted to 7 with0.1 N HCl and the mixture was subsequently lyophilized and further usedwithout purification. LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm,A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile,10-40% B over 10 min) t_(R)=6.8 min (LSD signal), m/z=340.2 for [M+H]⁺.¹H NMR (CD₃OD, 400 MHz) δ=1.34-1.42 (m, 8H, 2×OCH₂CH₃ and CH₂); 1.67 (m,2H, CH₂); 1.80 (m, 2H, CH₂); 2.20 (t, 2H, CH₂, ³J=7.6 Hz); 3.16 (s, 6H,N(CH₃)₂); 3.40 (m, 2H, CH₂); 3.72 (brs, 2H, CH₂); 4.18 (dq, 4H,2×CH₂CH₃, ³J(H,H)=7.0 Hz, ³J(H,P)=8.0 Hz); 4.50 (brs, 2H, CH₂). ¹³C NMR(CD₃OD, 100.5 MHz) δ=16.4 (d, 2×OCH₂CH₃, ³J=6.8 Hz); 23.2 (CH₂); 26.5(CH₂); 27.1 (CH₂); 37.9 (CH₂); 52.0 (CH₃); 62.1 (d, CH₂, ²J=4.6 Hz);64.5 (CH₂); 66.1 (d, CH₂, ²J=6.1 Hz), 66.8 (CH₂); 181.3 (CO).

[2-(diethoxy-phosphoryloxy)-ethyl]-dimethyl-[5(4-nitro-phenoxycarbonyl)-pentyl]-ammoniumtrifluoroacetate. To a solution of the carboxylic acid (26 mg, 0.076mmol) in dry acetonitrile (3 mL) were consecutively added triethylamine(0.01 mL, 0.084 mmol), p-nitrophenyl chloroformate (31 mg, 0.15 mmol)and DMAP (1 mg). The reaction was stopped after 4 h by addition of 0.1%TFA in water and subsequently purified by rp HPLC (Supelco DiscoveryC18, 21.2 mm×15 cm, 5 μm, A: 5% IPA, 95% H₂O, 0.1% TFA, B: 90% IPA, 10%H₂O, 0.1% TFA, 20 to 40% B over 12 min, t_(R)=10.3 min) to give thep-nitrophenyl ester as colorless oil (10 mg, 0.022 mmol, 28%). LC-MS(Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid in water,B: 0.1% formic acid in acetonitrile, 10-90% B over 8 min) t_(R)=4.3 min(UV signal), m/z=461.5 for [M+H]⁺. ¹H NMR (CDCl₃, 300 MHz) δ=1.34 (t,6H, 2×OCH₂C₃ ³J=7.2 Hz); 1.48 (m, 2H, CH₂); 1.81 (m, 4H, 2×CH₂); 2.63(t, 2H, CH₂, ³J=7.2 Hz); 3.27 (s, 6H, N(CH₁₃)₂); 3.51 (m, 2H, CH₂); 3.92(m, 2H, CH₂); 4.14 (dq, 4H, 2×CH₂CH₃, ³J(H,H)=7.5 Hz, ³J(H,P)=7.5 Hz);4.47 (m, 2H, CH₂); 7.27 (d, 2H, ³J=9 Hz, Phe-H); 8.25 (d, 2H, ³J=9 Hz,Phe-H). ¹³C NMR (CDCl₃, 75.4 MHz) δ=16.0 (d, 2×OCH₂CH₃, ³J=6.6 Hz); 22.4(CH₂); 23.8 (CH₂); 25.4 (CH₂); 33.6 (CH₂); 51.6; 60.8 (d, CH₂, ²J=4.8Hz); 63.3 (?); 64.7 (d, CH₂, ²J=5.9 Hz); 65.7; 122.5 (2× Phe-CH); 125.2(2×Phe-CH); 145.3 (Phe-C); 155.3 (Phe-C); 170.8 (CO).

Dimethyl-[5-(4-nitro-phenoxycarbonyl)-pentyl]-(2-phosphonooxy-ethyl)-ammoniumtrifluoro acetate. The protected p-nitrophenyl ester (20 mg, 0.043 mmol)was dissolved in dry acetonitrile (3 mL) and trimethyl silylbromide(0.11 mL, 0.87 mmol) was added. The reaction mixture was stirred at roomtemperature for 24 h. After adding 0.1% TFA in water (1 mL) the solutionwas purified by rp HPLC (Supelco Discovery C18, 21.2 mm×15 cm, 5 μm, A:0.1% TFA in water, B: 0.1% TFA in acetonitrile, 17 to 30% B over 14 min,t_(R)=12.7 min) to give the phosphocholine derivativePC₆-pNitrophenylester (4.6 mg, 0.011 mmol, 25%) as TFA salt. LC-MS(Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid in water,B: 0.1% formic acid in acetonitrile, 10-90% B over 10 min) t_(R)=3.8 min(UV signal), m/z=405.5 for [M+H]⁺.

(10-Carboxy-decyl)-[2-(diethoxy-phosphoryloxy)-ethyl]-dimethyl-ammoniumtrifluoro acetate

Fmoc-undecanoic acid (635 mg, 1.5 mmol) was dissolved in dry DMF (2 ml)and a solution of diisopropyl carbodiimid (0.12 ml, 0.75 mmol) in drydichloromethane (10 ml) was added. The resulting yellow mixture wasstirred at 0° C. for 20 min. After removal of the volatiledichloromethane the solution of the symmetrical anhydride was added tothe Wang resin (250 mg, 1.2 mmol/g, 0.3 mmol). DMAP (18 mg, 0.15 mmol)was added and the reaction mixture was stirred over night (W. C. Chan,P. D. White in Fmoc Solid Phase Peptide Synthesis, A Practical Approach(Eds.: W. C. Chan, P. D. White) Oxford University Press, Oxford 2000,pp. 55-56.) The resin was washed with DMF (5×2 min), dichloromethane(5×2 min) and was shrunk with diethyl ether. The procedure was repeatedone time and the final substitution level of 0.8 mmol/g (66%) wasdetermined based on UV spectrophotometry of the Fmoc-piperidine adduct.(ibid., pp 62-63)

After Fmoc deprotection with 20% piperidine/DMF the resin was swelled inTHF. A mixture of THF (2 ml), formaldehyde (40%, 0.7 ml, 1 mmol) andacetic acid (0.7 ml) were added and the resin was incubated for 5 min.Then sodium cyanoborohydrid (62 mg, 1 mmol) was added and the resin wasstirred at room temperature over night. Afterwards the resin was washedsuccessively with THF, H₂O and MeOH.

E. G. Brown, J. M. Nuss Tetrahedron Lett. 1997, 38, 8457-8460.

The resin was swelled in DMF and phosphoric acid diethylester-(2-bromo-ethyl ester)/phosphoric acid 2-bromo-ethyl ester diethylester (520 mg, 2 mmol) and DIEA (0.03 ml, 0.2 mmol) were added and thereaction mixture was heated to 60° C. for 4 d. The resin was washed withDMF, DCM and was shrunk with MeOH.

J. Cai, B. Wathey Tetrahedron Lett. 2001, 42, 1383-1385.

The product was finally cleaved from the resin by stirring it with amixture of TFA/H₂O/TIS (95:2.5:2.5) for 2 h. The carboxylic acid wasused after evaporation of the organic solvents, addition of water andlyophilization without further purification.

[2-(Diethoxy-phosphoryloxy)-ethyl]-dimethyl-[10-(4-nitro-phenoxycarbonyl)-decyl]-ammoniumtrifluoro acetate

To a solution of the carboxylic acid X in dry acetonitrile (5 ml) wereconsecutively added triethylamine (0.03 ml, 0.2 mmol), p-nitrophenylchloroformate (80 mg, 0.39 mmol) and DMAP (7 mg). The reaction wasstopped after 4 h by addition of 0.1% TFA in water and subsequentlypurified by RP HPLC (Supelco Discovery C18, 21.2 mm×15 cm, 5 μm, A: 0.1%TFA in IPA/H₂O 5:95, B: 0.1% TFA in IPA/H₂O 90:10, 15 to 45% B over 20min, t_(R)=16.5 min) to give the p-nitrophenyl ester X as colorless oil(4 mg, 0.007 mmol, %).

LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid inwater, B: 0.1% formic acid in acetonitrile, 10-90% B over 10 min)t_(R)=5.2 min (UV signal); M=531.5 (M⁺).

Dimethyl-[10-(4-nitro-phenoxycaxbonyl)-decyl]-(2-phosphonooxy-ethyl)-ammoniumtrifluoro acetate

The protected p-nitrophenyl ester (4 mg, 0.007 mmol) was dissolved indry acetonitrile (3 ml) and trimethyl silylbromide (0.11 ml, 0.07 mmol)was added after 0, 12 and 24 hours resp. The reaction mixture wasstirred at room temperature for totally 36 h. After adding 0.1% TFA inwater (1 ml) the solution was purified by rp HPLC (Hichrom C8, 21.2mm×25 cm, 10 μm, A: 0.1% TFA in ACN/H₂O 10:90, B: 0.1% TFA in ACN/H₂O90:10, 40 to 60% B over 15 min, t_(R)=14.2 min) to give thephosphocholine derivative (1.1 mg, 0.002 mmol, 27%) as TFA salt.

LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid inwater, B: 0.1% formic acid in acetonitrile, 10-90% B over 10 min)t_(R)=5.9 min (UV signal), M=475.4 (M⁺).

PC4

4-dimethylamino butyric acid methyl ester

same procedure as for PC6

¹H NMR (D₂O, 400 MHz) δ=2.06 (m, 2H, CH₂); 2.56 (t, 2H, ₃ J=7.2 Hz,CH₂); 2.93 (s, 6H, N(CH₃)₂); 3.22 (m, 2H, CH₂); 3.75 (s, 3H, COOCH₃).

¹³C NMR (CD₃OD, 100.5 MHz) δ=20.9; 31.2; 43.5; 52.3; 58.1; 174.3

LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid inwater, B: 0.1% formic acid in acetonitrile, 10-50% B over 10 min)t_(R)=1.3 min (ELSD signal); M=146.4 ((M+H)⁺).

[2-Diethoxy-phosphoryloxy)-ethyl]-(3-methoxycarbonyl-propyl)-dimethylammoniumtrifluoroacetate

The methyl ester X (516 mg, 3.5 mmol) was dissolved in dry acetonitrile(8 ml) and K₂CO₃ (484 mg, 3.5 mmol) was added. This suspension wasrefluxed under nitrogen gas for 24 h. After cooling to room temperaturewater was added and the pH was adjusted to 2 by adding TFA. The solutionwas purified by rp HPLC (Supelco Discovery C18, 21.2 mm×15 cm, 5 μm, A:0.1% TFA, 5% IPA in H₂O, B: 0.1% TFA, 10% H₂O in IPA, 0 to 10% B over 10min, t_(R)=8.8 min) and lyophilized to give the TFA salt of thequaternary amine as light yellow oil.

¹H NMR (CD₃OD, 400 MHz) δ=1.36 (t, 6H, 2×OCH₂CH₃, ³J=6.8 Hz); 2.08 (m,2H, CH₂); 2.47 (t, 2H, CH₂, ³J=7.2 Hz); 3.20 (s, 6H, N(CH₃)₂); 3.45 (m,2H, CH₂); 3.70 (s, 3H, COOCH₃); 3.76 (t, 2H, CH₂, ³J=4.4 Hz); 4.18 (dq,4H, OCH₂CH₃ ³J(H,H)=7.2 Hz, ³J(H,H)=7.2 Hz); 4.52 (bs, 2H, CH₂).

¹³C NMR (CD₃OD, 100.5 MHz) δ=16.4 (d, 2×OCH₂CH₃, ³J=6.1 Hz); 19.0 (CH₂);30.7 (CH₂); 52.3 (CH₃); 52.4 (CH₃); 62.1 (d, CH₂, ²J=4.5 Hz); 64.4(CH₂); 65.4 (CH₂); 66.1 (d, CH₂, ²J=6.0 Hz); 174.0 (CO).

(3-carboxy-propyl)-[2-(diethoxy-phosphoryloxy)-ethyl]-dimethylammoniumtrifluoroacetate

same procedure as for PC6

LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid inwater, B: 0.1% formic acid in acetonitrile, 10-50% B over 10 min)t_(R)=1.8 min (ELSD signal); M=312.3 (Mt).

[2-(Diethoxy-phosphoryloxy)-ethyl]-dimethyl-[3-(4-nitro-phenoxycarbonyl)-propyl]-ammoniumtrifluoroacetate

To a solution of the carboxylic acid in dry acetonitrile (5 ml) wereconsecutively added triethylamine, p-nitrophenyl chloroformate and DMAP.The reaction was stopped after 4 h by addition of 0.1% TFA in water andsubsequently purified by RP HPLC (Supelco Discovery C18, 21.2 mm×15 cm,Sun, A: 0.1% TFA, 5% IPA in H₂O, B: 0.1% TFA 10% H₂O in IPA, 10 to 30% Bover 13 min, t_(R)=11.8 min) to give the p-nitrophenyl ester ascolorless oil.

LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid inwater, B: 0.1% formic acid in acetonitrile, 10-90% B over 10 min)t_(R)=3.1 min (UV signal); M=433.2 (M⁺).

Alternative Scheme for Preparation of PC6

During the priority year, an alternative strategy for preparing thephosphocholine ligand PC6 was developed. This is outlined below.

This strategy for preparation of PC6 (compound 9) is outlined in scheme3.

Phosphorylation was chosen as the last step, since this function wasconsidered troublesome, even in a protected form, to carry through othernecessary transformation steps.

Commercially available aminohexanoic acid 2 was N,N-dimethylated¹¹ ¹² byan Eschweiler-Clarke-type procedure (treatment with formaldehyde andhydrogen/palladium/carbon), and the resulting product (3, 86%) wasesterified by treatment with methanolic hydrogen chloride to give themethyl ester 4 (96%). Quartenarization was effected by reacting 4 withmonomethoxytrityl-protected bromoethanol 1, forming derivative 5 (73%).Unprotected bromoethanol could also be reacted with 4, but the reactionyield was lower and the product was more difficult to detect and purify.Monomethoxytrityl protection conferred lipophilicity and TLCdetectability (UV visible and acid stainable) to the crucialintermediate 5. Saponification of 5 with lithiumhydroxide/water/t-butanol gave the corresponding acid 6 (95%), which wastreated without isolation with p-nitrophenol and diisopropylcarbodiimide in pyridine to give the p-nitrophenyl ester 7 (86%). Brieftreatment of 7 with formic acid gave the corresponding alcohol 8, whichwas used directly in the next step, because of its sensitivity toself-condensation. Reaction of crude 8 at 0° C. in acetonitril withfirst an excess of phosphorous oxychloride and triethylamine, then watergave the desired monophosphate 9 in 22% yield from 7, after HPLCpurification. Compound 9 gave the expected NMR and LC-MS spectra, andwas stable in aqueous solution at acidic pH (0.1% TFA). For convenientstorage, however, the material was kept as a DMSO stock solution in thefreezer.

General methods—Concentrations were performed at reduced pressure (bathtemperature <40° C.). NMR spectra were recorded at 298° K with a VarianUnity 500 MHz spectrometer. Only selected NMR data are reported.Assignments were corroborated by appropriate 2-D experiments.Electrospray mass spectroscopy (ES-MS) for dilute methanol oracetonitril solutions was performed in the direct-inlet mode using aPerkin-Elmer SCIEX API 150-EX mass spectrometer. The positive-ion modespectra were recorded and processed using the manufacturer software onan Apple MacIntosh Quadra computer.

TLC was performed on Silica Gel F254 (Merck, Darmstadt, Germany) plateswith detection by UV-light and/or by staining with either 5% ninhydrinin butanol or 5% sulfuric acid in ethanol. Column chromatography wasperformed on Matrex silica gel 60 Å (35-70 μm, Amicon) unless otherwisestated. Preparative HPLC was carried out using a Varian HPLC systemconsisting of a 9012 gradient pump, a 9065 diode array detector, and aPC computer system running the Varian Star HPLC software. Isolute C-18EC reversed phase silica (40-70 micron particle size) from InternationalSorbent Technology Ltd (Mid Glamorgan, UK) was packed and eluted withthe specified solvents in open glass columns. Other reagents andsolvents were purchased with high commercial quality and were usedwithout further purification unless otherwise stated.

2-Monomethoxytrityloxy-1-bromoethanol (1)—This compound was preparedanalogously to the corresponding trityl derivative¹³. Briefly,monomethoxytrityl chloride (3.09 g, 10 mmol) was added to a solution ofbromoethanol (1.0 g, 8.0 mmol) in dry pyridine (5 mL) at roomtemperature. After 12 hrs, TLC (hexane-ethyl acetate, 9:1) indicatedpresence of two UV-absorbing spots, one (major) with rf 0.9, one (minor)with rf 0.7. Water (0.1 mL) was added, and after 15 min stirring at r.t.the mixture was partitioned between ethyl acetate and aq 2M sulfuricacid, the organic layer was washed with aq. sodium bicarbonate andconcentrated. The residue was purified by column chromatography onsilica gel (160 g) packed in pentane-ethyl acetate 95:5 and eluted witha pentane-ethyl acetate 95:5-90:10 stepwise gradient. Collection of themain band gave 1 as a syrup (2.75 g, 87%), which crystallized onstanding. Alternatively, chromatography could be omitted and the crudesyrup crystallized directly from methanol to give pure crystals in ayield of 40-50%. Direct-inlet ES-MS showed a large peak at m/z 273(monomethoxytrityl cation) but only two very small peaks at 419/421(M+Na). H-NMR data (CDCl₃): 3.44/3.48 (2 multiplets, 2+2H, OCH₂CH₂Br),3.83 (s, 3H, ArOCH3), 6.88 (d, 2H, ArH), 7.24-7.52 (m, 12H, Ar—H).

6-(N,N-Dimethylamino)-N-[2-(monomethoxytrityloxy)-ethyl]-hexanoic acidmethyl ester (5)—

A solution of 6-aminohexanoic acid (2, 13.2 g) in aqueous formaldehyde(37%, 40 mL) was mixed with Pd/C (1.0 g) and the mixture washydrogenated in a Parr stainless steel apparatus with magnetic stirringovernight at room temperature and 50 bar. A check by TLC (ethylacetate-methanol-acetic acid-water, 6:3:3:2, ninhydrin detection)revealed almost complete conversion from starting material (rf 0.7) to aslower-migrating product (rf 0.5, browner color). The reaction mixturewas filtered through a bed of Celite, the filter bed was washed withwater (20 mL), diluted (60 mL), and the solution was slowly passedthrough a column of Dowex-50 Wx2-100 mesh (H+ form, 0.7 meq/mL, 150 mL,carefully pre-washed with milli-Q water). The column was then washedwith milli-Q water (200+100 mL), and then the product was eluted with 2%aq ammonia (400 mL). The eluate was monitored by TLC (ethylacetate-methanol-acetic acid-water, 6:3:3:2, ninhydrin detection). Theappropriate fractions were partially evaporated, then lyophilized toleave a semi-solid residue (13.7 g, 86%). Direct inlet ES-MS revealed astrong peak at m/z 160.2 (M+H), corresponding to the N,N-demethylatedproduct (3).

A fraction of this crude material (3.97 g, 25 mmol) in methanol (75 mL)was cooled in ice and stirred while thionyl chloride (10 mL, 134 mmol)was added dropwise during 30 min, after which the cooling was removedand the mixture was left at room temperature overnight. A check by T/LC(ethyl acetate-methanol-acetic acid-water, 10:3:3:2, ninhydrindetection) revealed conversion to a slightly faster-moving compound withweaker staining and a different tint. There were also minorfaster-moving impurities carrying over from the previous step. After 16hrs, the reaction mixture was concentrated and co-concentrated severaltimes with methanol to give a residue (5.05 g) which crystallizedpartially on standing. Direct-inlet ES-MS of this material revealed astrong peak at m/z 174.0 (M+H), corresponding to6-(N,N-dimethylamino)-hexanoic acid methyl ester (4).

A fraction of this crude material (1.12 g, purity approximately 75%, 4.0mmol), dry acetonitril (25 mL), 2-(monomethoxytrityloxy)-2-bromoethanol(1, 1.60 g, 4.0 mmol), and solid anhydrous sodium carbonate (800 mg) wasrefluxed (bath temp 70° C.) overnight, after which TLC showed onlypartial conversion, so more 1 (800 mg, 2.0 mmol) and sodium carbonate(500 mg) was added, and reflux was continued for another 24 hrs. TLCthen showed that the majority of starting material had disappeared, andthat there was a new, faster-migrating, ninhydrin-staining andTV-absorbing spot. The mixture was partitioned between dichloromethaneand water (the desired material was in the organic phase), the aq. phasewas washed with a little dichloromethane, and the combined organiclayers were washed with a little water, then concentrated to a smallvolume (10 mL), which was partitioned between methyl-t-butyl ether andwater (the desired material was now in the aqueous phase). The organicphase was washed with a little water, and the combined aqueous layer waswashed with a little methyl-t-butyl ether. The combined aqueous solutionwas rotavapored shortly to remove non-aqueous solvents (approx. ⅓ ofvolume was removed) and the residual solution was passed through aBond-Elut C-18-EC column (35 g. packed in methanol and then equilibratedwith water), washed after with water, eluted with first 20%, 50 and thenfinally 60% methanol in water. The desired material eluted as a pureband with the 60% methanol eluent. Appropriate fractions were collected,a few drops of pyridine were added, and the solution was lyophilized,leaving behind compound 5 as a white solid (1.53 g, 73%). ES-MS showed astrong m/z 490 peak (M+). H-NMR data (CDCl3): 1.31 (m, 2H, H-4), 1.65(m, 2H, H-3), 1.75 (m, 2H, H-5), 2.31 (t, 2H, H-2), 3.30 (s, 6H,(CH3)2N), 3.47 (m, 2H, H-6), 3.62 (m, 2H, OCH2CH2N), 3.68 (s, 3H,COOCH3), 3.82 (s, 3H, ArOCH3), 3.93 (m, 2H, OCH2CH2N), 6.89 (d, 2H,ArH), 7.24-7.41 (m, 12H, ArH).

6-(N,N-Dimethylamino)-N-[2-(monomethoxytrityl oxy)-ethyl]-hexanoic acidp-nitrophenyl ester (7)—A solution of compound 5 (420 mg, 0.86 mmol) in2:1-butanol-water (6.0 mL) was mixed with lithium hydroxide (72 mg) andthe solution was stirred at room temperature for 3 h, after which TLC(ethyl acetate-methanol-acetic acid-water, 12:3:3:2) showed completeconversion into a compound with a slightly lower rf., An ES-MS spectrumat this stage showed m/z 476.0 and 482.3 peaks (M+H and M+Li,respectively). The mixture was diluted with water (10 mL), the pH wasadjusted to 7.5 with aq 0.1 M hydrochloric acid, and then the solutionwas lyophilized to give crude 6 (510 mg, contaminated with lithiumchloride). This material (approximately 0.86 mmol) was dissolved in indry pyridine (6.0 mL), and then p-nitrophenol (400 mg, 2.86 mmol), anddiisopropylcarbodiimide (270 microL, 0.176 mmol) was added. Afterstirring for 3 hrs at RT another portion of diisopropyl carbodiimide(200 microL) was added, and the mixture was stirred for another 24 hrsat r.t., after which TLC (ethyl acetate-methanol-acetic acid-water,15:3:3:2) indicated complete reaction. The mixture was evaporated andcoevaporated ×3 with dichloromethane-toluene to remove pyridine. Theresidue was partitioned between aqueous 0.2 M triethylammonium acetate(pH 5.0) and methyl t-butyl ether (about 100 mL of each solvent andheavy shaking was necessary to dissolve everything). TLC (ethylacetate-methanol-acetic acid-water, 10:3:3:2, UV and ninhydrindetection) showed that the product was exclusively in the aq. phase,whereas most of the p-nitrophenol and also other UV-absorbing impuritieswere in the organic phase. The organic phase was washed with a littleaq. buffer, and the combined aqueous phases were washed with methylt-butyl ether, then extracted with dichloromethane (TLC now indicatedcomplete transfer of product into the organic phase). The aqueous layerwas washed with a little dichloromethane, and the combined organiclayers were washed with a small amount of buffer, and carefullyseparated. At this, TLC revealed presence of a very pure material, andH-NMR also confirmed this. The material was kept in the freezer insolution, but could, if necessary, be concentrated to give syrupy, crude7 (440 mg, 86%). In this form, the material also remain unchanged forweeks at freezer temperature. An ES-MS of the material showed a strongsignal at m/z 596.8 (M+).

H-NMR data (CDCl3): 1.41 (m, 2H, H-4), 1.75-1.85 (m, 4H, H-3, H-5), 2.63(t, 2H, H-2), 3.24 (s, 6H, (CH3)2N), 3.49 (m, 2H, H-6), 3.62 (m, 2H,OCH2CH2N), 3.81 (s, 3H, ArOCH3), 3.84 (m, 2H, OCH2CH2N), 6.89 (d, 2H,ArH), 7.24-7.41 (m, 12H, ArH), 8.28 (d, 2H, ArH).

6-(N,N-Dimethylamino)-N-[(2-phosphoryloxy)-ethyl]-hexanoic acidp-nitrophenyl ester (9)—A solution of compound 7 (55 mg, 0.10 mmol) wasdissolved in formic acid (2 mL) and kept at RT for 60 min, then it wasconcentrated and co-concentrated ×3 with dry acetonitril. The residualcompound 8, showing a reasonable purity by TLC (ethylacetate-methanol-acetic acid-water, 6:3:3:2, ninhydrin and UV detection)and ES-MS (m/z 325.2, M+), was dissolved in dry acetonitril (0.5 mL) andadded to a cooled (0° C.) solution of phosphorous oxychloride (0.019 mL,0.2 mmol, freshly distilled) and triethylamine (0.069 mL, 0.5 mmol) indry acetonitril (0.5 mL). After 1 h at 0° C., more phosphorousoxychloride (0.05 mL) and triethylamine (0.05 mL) was added, and afteranother hour water (0.05 ml) was added and the mixture was furtherstirred at 0° C. for 1 hour, after which TLC showed disappearance ofmost starting material, and appearance of a UV-absorbing spot with verylow (rf 0.05) mobility. The reaction mixture was diluted with 0.2% aqTFA (3.8 mL), washed with methyl t-butylether (3.5 mL), the aq phase wascarefully separated, rotavapored shortly at 35° C. (down to approx. ⅔volume) and the resulting solution was injected in 400 microL portionson a Supelco Discovery C-18 column (15×21.2 cm, 5 microM), using a flowof 8.0 mL/min and a gradient over 40 min from 82% to 58% water inacetonitril (a 0.1% TFA concentration was maintained throughout). Onemajor and one minor UV-absorbing (300 nm) peak were seen at 18-20 minand 24-26 min respectively, corresponding to desired product 9 andstarting material 8, respectively. Fractions containing 9 were pooled,concentrated to a small volume, and lyophilized to give pure 9 as anamorphous material (9 mg, 22% from 7). An LC-MS spectrum showed adominating m/z 405.2 signal (M+). H-NMR data (DMSO-d6): 1.38 (m, 2H,H-4), 1.67-1.78 (m, 4H, H-3, H-5), 2.69 (t, 2H, H-2), 3.01 (s, 6H,(CH3)2N), 3.35 (m, 2H, H-6), 3.60 (m, 2H, OCH2CH2N), 4.24 (m, 2H,OCH2CH2N), 7.44 (d, 2H, ArH), 8.31 (d, 2H, ArH). H-NMR data (D2O,HOD=4.68): 1.35 (m, 2H, H-4), 1.66 (m, 2H, H-3 or H-5), 1.72 (m, 2H, H-3or H-5), 2.59 (t, 2H, H-2), 3.02 (s, 6H, (CH3)2N), 3.28 (m, 2H, H-6),3.52 (m, 2H, OCH2CH2N), 4.19 (m, 2H, OCH2CH2N), 7.22 (d, 2H, ArH), 8.19(d, 2H, ArH).

Peptide Synthesis

A library of sixteen 42-residue peptides (FIG. 1) was prepared bysolid-phase peptide synthesis. The components of the library varied withregards to the number of charged residues and the position ofincorporation of the ligand. The synthesis procedure was the same ineach case and is exemplified here by that of 3-C15L8Cys24 (TA4Cys(Acm)).

The polypeptide was synthesized on a Pioneer automated peptidesynthesizer (Applied Biosystems) using standard Fmoc chemistry. Thesynthesis was performed on 0.1 mmol scale on Fmoc-PAL-PEG-PS resin witha substitution level of 0.2 mmol/g. The following amino acid derivatives(Novabiochem AG) were used: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH,Fmoc-Leu-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Nle-OH,Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Val-OH. The side chain of Lys 15 was Alocprotected, whereas Boc protection was used for Lys 8. A fourfold excessof amino acid derivative was used for each coupling step. TBTU(O-(7-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate,0.5M in DMF) was used as coupling reagent in combination with DIEA(diisopropyl ethyl amine, 1M in DMF). A standard coupling time of 60 minwas used, except for Gln, Cys, Pro and His (90 min) and the firstresidue, Arg, and Asn (2 h). After the conventional automated solidphase peptide synthesis was completed, the free N-terminus wasacetylated with acetic anhydride treating the resin for 2 h with amixture of acetic anhydride/dichloromethane (3:1) and the resin waswashed with dichloromethane (6×) and shrunk with diethyl ether. The Alocprotecting group was removed with 3 eq. oftetrakis(triphenyl)palladium(0) in chloroform/acetic acid/NMM (N-methylmorpholine (18:2:1) for 2.5 h. The resin was consecutively washed withDIEA (0.5% in DMF), sodium diethyl dithiocarbamic acid (0.5% in DMF),DMF, dichloromethane and shrunk with diethyl ether.7-methoxycoumarin-3-carboxylic acid (3 eq.) was coupled to the sidechain of Lys 15 with HBTU (3 eq.), HOBt (3 eq.) and DIEA (6 eq.) in DMFfor 4 h. The resin was washed with DMF, dichloromethane and shrunk withdiethyl ether. The peptide was cleaved from the resin with TFA, TIS(triisopropyl silane) and water (95:2.5:2.5, 1 ml per 100 mg of resin)over 2 h at room temperature. The TFA was evaporated under reducedpressure and the peptide was precipitated by addition of cold diethylether, centrifuged, washed and lyophilized. The crude product waspurified by RP HPLC (Hichrom C8, 21.2 mm×25 cm, 10 μm, A: 0.1% TFA, 10%ACN in H₂O, B: 0.1% TFA 10% H₂O in ACN, 38 to 46% B over 18 min,t_(R)=16.0 min).

Preparation of Peptide Scaffold Dimers.

Disulfide bond formation (H. Tamamura, A. Otaka, J. Nakamura, K. Okubo,T. Koide, K. Ikeda, T. Ibuka, N. Fuji Int J. Peptide Protein Res. 1995,45, 312-319.):

The peptide (2.4 mg (0.51 μmol) of 3-C15L8Cys(Acm)24) was dissolved inTFA (200 μl) and silver triflate (5 mg, 20 mmol) and anisole (1 drop)were added. The reaction mixture was incubated in the refrigerator at 4°C. for 24 h. After addition of diethyl ether the peptide precipitatedand was washed 3 times with cold diethyl ether. To the precipitate wereadded DMSO (0.25 ml) and 1 N HCl (1 ml). The reaction mixture wasstirred at room temperature for 22 h. The suspension was centrifuged andthe precipitate was discarded. The liquid was neutralized with dilutedNaOH and analyzed by RP-HPLC (analytical run: Varian C18, 150×4.6 mm, 5μm, A: 0.1% TFA, 10% ACN in H₂O, B: 0.1% TFA 10% H₂O in ACN, 30 to 60% Bover 30 min, t_(R)=23.4 min.

Reaction of PC₆-pNitrophenylester with TA₄ ₊ Cys(Acm).

This is a specific example of a protocol for incorporating aphosphocholine derivative into a peptide, in this case TA₄ ₊ with aprotective group Acm on the Cys-residues.

To a solution of TA₄ ₊ Cys(Acm) peptide (500 μg, 0.1 μmole) in 100 μL of0.1 M phosphate buffer pH 8 were added 2.8 μL of 0.1 MPC6-pNitrophenylester in DMSO (0.3 μmole, 3 eq.). The reaction mixturewas incubated at 4° C. LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm,A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile,10-90% B over 10 min) t_(R)=4.1 min (UV signal), m/z=670.5 for [M+8H]⁸⁺,766.1 for [M+7H]⁷⁺, 893.3 for [M+6H]⁶⁺, 1071.9 for [M+5H]⁵⁺.

Immobilization of TA4+PC6Cys(Acm) Peptide on Porous Polystyrene Coatedwith F108-2-Pyridyl Disulfide

To a solution of TA₄ ₊ PC6Cys(Acm) peptide (90 μg, 18 nmol) in 18 μL of0.1 M phosphate buffer pH 8 were added 1.8 μL of 5 mg/mL iodine in 50%MeOH in water (8.6 μg, 34 nmol, 2-fold excess relative to thiol groups).The reaction mixture was incubated at 25° C. for 20 min, cleaned withPepClean C18 Columns (Pierce) and added directly to a 50 μL suspensioncontaining 10 mg of F108-PDS-coated porous particles in phosphatebuffer.

Kinase-Peptide Conjugation

To a solution of TA₄ ₊ PC6Cys(Acm) peptide (90 μg, 18 nmol) in 18 μL of0.1 M phosphate buffer pH 8 were added 1.8 μL of 5 mg/mL iodine in 50%MeOH in water (8.6 μg, 34 mmol, 2-fold excess relative to thiol groups).The reaction mixture was incubated at 25° C. for 20 min, cleaned withPepClean C18 Columns (Pierce) and added directly to a 1.5 mL of 1.3mg/ml Kinase-PDS in phosphate buffer.

Formation of TA₄PC6CysH

Reaction of PC6-pNitrophenylester with TA₄Cys(Acm). To a solution ofTA₄Cys(Acm) peptide (1 mg, 0.2 μmol) in 1 mL of 0.1 M phosphate bufferpH 8 were added 10 μL of 0.1 M PC6-pNitrophenylester in DMSO (0.8 μmol,5 eq.). The reaction mixture was incubated at 4° C. for 2 days, purifiedby gel filtration using NAP-10 columns (GE Healthcare) to have 1.55 mLof TA₄PC6Cys(Acm) in water and finally lyophilized. LC-MS (PhenomenexGemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid in water, B: 0.1%formic acid in acetonitrile, 10-90% B over 10 min) t_(R)=4.1 min (UVsignal), m/z=856.0 for [M+6H]⁶⁺, 1026.8 for [M+5H]⁵⁺, 1282.6 for[M+4H]⁴⁺.

Cysteine deprotection. TA₄PC6Cys(Acm) was dissolved in 1 mL ofTFA/anisole (99:1) and 6 mg of AgOTf were added (20 μmol, 100 eq.).After 2 hours at 4° C., the peptide silver salt was precipitated withcold ether, isolated by centrifugation and washed 2 times with ether.116 μL of 10 mg/ml DTT in 1 M acetic acid were added and the solutionvolume was extended to 1 mL with 1 M acetic acid. After one night at 25°C. the peptide was purified by gel filtration using NAP-15 columns tohave 1.5 mL of TA4PC6CysH in water and lyophilized.

LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid inwater, B: 0.1% formic acid in acetonitrile, 10-90% B over 10 min)t_(R)=4.6 min (UV signal), m/z=843.5 for [M+6H]⁶⁺, 1012.3 for [M+5H]⁵⁺,1264.6 for [M+4H]⁴⁺.

Formation of TA₄ ₊ PC6CysH

Reaction of PC₆-pNitrophenylester with TA₄ ₊ Cys(Acm). To a solution ofTA₄ ₊ Cys(Acm) peptide (1 mg, 0.2 μmol) in 1 mL of 0.1 M phosphatebuffer pH 8 were added 10 μl′ of 0.1 M PC6-pNitrophenylester in DMSO(0.8 μmol, 5 eq.). The reaction mixture was incubated at 4° C. forovernight, purified by gel filtration using NAP-10 columns (GEHealthcare) to have 1.5 mL of TA₄ ₊ PC6Cys(Acm) in water and finallylyophilized. LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1%formic acid in water, B: 0.1% formic acid in acetonitrile, 10-90% B over10 min) t_(R)=4.0 min (UV signal), m/z=670.3 for [M+8H]⁸⁺, 766.1 for[M+7H]⁷⁺, 893.4 for [M+6H]⁶⁺.

Cysteine deprotection. TA₄ ₊ PC6Cys(Acm) was dissolved in 1 mL ofTFA/anisole (99:1) and 6 mg of AgOTf were added (20 μmol, 100 eq.).After 2 hours at 4° C., the peptide silver salt was precipitated withcold ether, isolated by centrifugation and washed 2 times with ether.116 μL of 10 mg/ml DTT in 1 M acetic acid were added and the solutionvolume was extended to 1 mL with 1 M acetic acid. After one night at 25°C. the peptide was purified by gel filtration using NAP-15 columns tohave 1.5 mL of TA₄ ₊ PC₆CysH in water and lyophilized.

LC-MS (Phenomenex Gemini, C18, 5 μm, 150×3.0 mm, A: 0.1% formic acid inwater, B: 0.1% formic acid in acetonitrile, 10-90% B over 10 min)t_(R)=4.6 min (UV signal), m/z=661.5 for [M+8H]⁸⁺, 755.9 for [M+7H]⁷⁺,881.6 for [M+6H]⁶⁺.

Incorporation of Ligand into Each Polypeptide Scaffold of the PeptideLibrary.

A more general protocol for incorporation of a phosphocholine derivativeis given below. A person skilled in the art will be able to adapt thisprotocol in order to optimize it for specific conditions that may apply.

Each peptide from the library is dissolved in 1 mL 50 mM phosphatebuffer, pH 8, to a concentration of 1 mM to form a stock solution. Theamount of peptide needed to form the stock solution is calculated fromits molecular weight assuming a water content of 25%. The concentrationof each stock solution is determined using an extinction coefficient of20300 M⁻¹ cm⁻¹ at 355 nm. 100 μL of each stock solution is transferredto a well of a microtitre plate and two eq. of the active ester of theligand to be tested dissolved in DMSO at an initial concentration of 100mM is added and left to react for 1 hr.

Affinity Measurements

In a separate set of wells of the microtitre plate the C-reactiveprotein (CRP) was dissolved to a concentration of 500 nM in 10 mM HEPES,150 mM NaCl and 5 mM CaCl₂ at pH 7.4. Each one of the polypeptidesfunctionalized with ligand and fluorophore was diluted and added to eachwell containing the target protein, to give a final concentration ofpeptide of 500 mM.

As a negative control, in a parallel experiment the same procedure wasapplied using polypeptides with fluorophore but without ligand. Themicrotitre plate was introduced into a fluorescence based plate readerset for excitation at 355 nm and emission at 420 nm. Peptides for whichthe fluorescence emission was different between polypeptidefunctionalized with ligand and polypeptide without ligand wereconsidered as “hits” and selected for more careful analysis.

An experimental protocol for careful analysis of affinity using afluorimeter is given in Table I and FIG. 2. The polypeptide used has thesequence named 3-C15L8. Although the analysis of the fluorescent data iscomplicated by the fact that C-reactive protein has five binding sitesfor phosphocholine it is clear that the affinity for the functionalizedpolypeptide in the presence of 5 mM CaCl₂ is compatible with adissociation constant of low nM or better.

The corresponding experiment with the polypeptide carrying fluorophorebut no ligand showed no change in fluorescence as a function of theconcentration of CRP, showing that the polypeptide scaffold itself hasvery low affinity for CRP. Consequently, the combination of polypeptideand phosphocholine derivative forms a binder for CRP where cooperativeinteractions between the components of the functionalized polypeptideand CRP gives rise to a very high overall affinity.

TABLE 1 Conc peptide Conc CRP Vol Vol Peptide Buffer (nM) (nM) CRPpeptide Vol buffer Intensities Average Error 4-C15L8- Bis Tris 20 mM, 5mM 100 0 0 10 490 456.78 486.4 527.63 490.27 35.58319 PC6 CaCl2 pH 7.2100 5 0.5 10 489.5 449.62 477.97 489.83 472.4733 20.66086 100 10 1 10489 456.17 455.1 450.21 453.8267 3.177489 100 25 2.5 10 487.5 446.88433.51 446.39 442.26 7.581682 100 50 5 10 485 423.14 393.56 409.77408.8233 14.81271 100 75 7.5 10 482.5 406.08 365.75 408.88 393.5724.13347 100 100 10 10 480 399.31 381.79 390.55 12.38851 100 150 15 10475 413.76 389.49 401.625 17.16148 100 200 20 10 470 405.77 405.774-C15L8 Bis Tris 20 mM, 5 mM 100 0 0 10 490 CaCl2 pH 7.2 100 5 0.5 10489.5 100 10 1 10 489 100 25 2.5 10 487.5 100 50 5 10 485 100 75 7.5 10482.5 100 100 10 10 480 100 150 15 10 475 100 200 20 10 470 — Bis Tris20 mM, 5 mM 0 0 0 0 500 CaCl2 pH 7.2 — Bis Tris 20 mM, 5 mM 0 1500 15 0485 CaCl2 pH 7.2Solid Phase CRP Detection Assay

One embodiment of a CRP detection assay having the binder of the presentinvention bound to a solid phase is schematically shown in FIG. 3.

In this assay the capture of CRP is accomplished by a CRP-binderattached to latex particles and the recognition signal is generated bythe binder conjugated to the enzyme pyruvate kinase. The kinasephosphorylates ADP, with the help of added PEP (phosphoenolpyruvate), toform ATP. The ATP, in turn, forms a complex with the enzyme luciferasewhich enables it to catalyze the oxidation of luciferin to oxyluciferin.This reaction generates light. The light intensity becomes a directmeasure of the amount of kinase in the sample, which in turn is ameasure of the amount of CRP bound.

Preparation of CRP-Binders Bound to Latex Particles

Monodisperse polystyrene latex particles with a diameter of 239 nm weresuspended in ultra-pure (MilliQ) water containing the polymericsurfactant Pluronic F108, containing pyridyl disulfide (PDS) conjugatedend-groups to allow linking of thiol containing ligands. The surfactantmolecules adsorb to the particles via their hydrophobic center blocks¹⁴.The number of Pluronic molecules taken up, through over-nightadsorption, by each particle was determined by sedimentation field-flowfractionation, SdFFF to be 16500¹⁵. The PDS groups were then replaced byeither the Cys-containing CRP-binder. A protocol for immobilising abinder (Ta4+) is given above. The analysis by SdFFF indicated that eachparticle had taken up 2560 binders.

The enzyme pyruvate kinase (type III from Rabbit muscle; Sigma-Aldrichproduct nr. P9136-5KU) was assayed, either as received, or aftertreatment with the bifunctional linker molecule, SPDP (PierceBiotechnology, product nr. 21857), or after attachment to the binder viaa disulfide bridge (exemplary protocol given above). The three forms ofthe enzyme were assayed for their respective ability to phosphorylateADP in the following manner: aliquots containing 0.36 mg kinase in 50 mMglycine-TRIS buffer of pH 7.6 were each added to the “luminescencereagent”, i.e. a mixture of 40 μL luciferase (“Thermostable”, fromBioThema AB, Stockholm) at 0.5 mg/mL, 10 μL PEP (Sigma-Aldrich) at 8.9mg/mL. 10□L 15 mM luciferin (Sigma-Aldrich). At time t=zero aliquots of10 μL ADP (Sigma-Aldrich, 2.6 mg/mL) were added to each sample mixtureand the light emission was followed with a CCD camera for 300 sec. Theresults are shown in FIG. 4. The three forms were largely of comparableactivity. The commercial product was unexpectedly somewhat less activethan its modified forms, probably caused by an (unintended) removal ofsome minor inhibitor during the processing.

The photon flux was measured by a CCD camera from a reaction mixture ofthe type illustrated in FIG. 3 and, for comparison, from a reactionmixture having an CRP-specific IgY antibody as CRP-binding agent insteadof the CRP-binder of the present invention. The reagent concentrationsand total amounts were the same as described for the “luminescencereagent” in FIG. 4. The samples were either CRP-free serum or serumcontaining 12 mg CRP/L. The highest photon flux was that provided by amixture wherein CRP-binders on the particle surfaces had captured CRPfrom the concentrated serum sample. After 3 cycles of washing andcentrifugation the composite sample was allowed to take up binder-kinaseconjugate; it was then mixed with the above luminescence reagent andallowed to generate its photon flux. Similarly, particles containingantibodies (IgY anti-CRP, a gift from Prof. A. Larsson, Uppsala AcademicHospital) were allowed to bind CRP from the serum of known (12 mg/L)concentration. An antibody-kinase conjugate was added to generate thephoton flux, as described. The CRP-free serum was used as blank. Inaddition to the four samples just described, there was one thatconsisted only of surfactant coated nanoparticles of the same size asthose carrying binders or antibodies in the four actual samples. Theresults are shown in FIG. 5. The figure clearly shows that thebinder-based detection of CRP gives a strong signal, while thebinder-based signal from CRP-free serum gives a straight baseline ofzero light intensity. The antibody-based detection gives a somewhatweaker signal in the CRP-containing serum, but demonstrates a highernon-specific adsorption than the binder-analogue to judge from thesignal resulting from the CRP-free serum. The surfactant-coatedparticles are clear blanks in this study.

An experiment analogous to that of FIG. 5 was performed onbinder-conjugated latex particles (10 μL, 10% w/v polystyrene particleswith 239 nm diameter). The particles were added to 100 μL of serum fromdog, horse, or human subjects where the former two had unknown contentsof CRP and the latter contained 12 mg/L. After 10 minutes of incubationthe particles were washed three times with centrifugation and thenincubated with binder-kinase conjugate for 15 minutes. After athree-fold wash procedure, and addition of the “luminescence reagent”described under FIG. 4, the samples were scanned by a luminometer fromBioThema which measures cumulative photon generation and computes ratesof ATP generation as a measure of kinase concentration, in turnproportional to the amount of CRP in the sample. The results are shownin FIG. 6 and Table 1. From this analysis it is clear that the bindershave the ability to capture CRP not only from humans, but from dog andhorse as well.

TABLE 2 Sample Reaction rate (pmol/min) Human CRP 0.045 ± 0.007 Dog CRP0.084 ± 0.009 Horse CRP 0.026 ± 0.007

REFERENCES

-   ¹ Hansen, H. J., LaFontaine, G., Newman, E. S., Schwartz, M. K.,    Malkin, A., Mojzisik, K., Martin, E. W., Goldenberg, D. M., (1989)    Solving the problem of antibody interference in commercial    “sandwich”-type immunoassays of carcinoembryonic antigen. Clin.    Chem., 35, 146-151.-   ² Moseley, K. R, Knapp, R. C., Haisma, H. J., (1988) An assay for    the detection of human anti-murine immunoglobulins in the presence    of CA125 antigen. J. Immunol. Methods, 106, 1-6.-   ³ R. P. Jain, R. M. Williams Tetrahedron 2001, 57, 6505-6509.-   ⁴ A. Kamitani, N. Chatani, S. Murai Angew. Chem. 2003, 115,    1435-1437; Angew. Chem. Int. Ed. 2003, 42, 1397-1399.-   ⁵ S. Kim, J. I. Lee, Y. C. Kim J. Org. Chem. 1985, 50, 560-565.-   ⁶ C. E. McKenna, M. T. Higa, N. H. Cheung, M.-C. McKenna Tetrahedron    Lett. 1977, 18, 155-158. b) C. E. McKenna, J. Schmidhauser J. Chem.    Soc., Chem. Comm. 1979, 739.-   ⁷ P. A. Bartlett, L. A. McQuaid J. Am. Chem. Soc., 1984, 106,    7854-7860.-   ⁸ W. C. Clan, P. D. White in Fmoc Solid Phase Peptide Synthesis, A    Practical Approach (Eds.: W. C. Chan, P. D. White) Oxford University    Press, Oxford 2000, pp. 55-56.-   ⁹ E. G. Brown, J. M. Nuss Tetrahedron Lett. 1997, 38, 8457-8460.-   ¹⁰ J. Cai, B. Wathey Tetrahedron Lett. 2001, 42, 1383-1385.-   ¹¹ R. P. Jain, R. M. Williams: Asymmetric synthesis of    (S)-(+)-carnitine and analogs, Tetrahedron 57 (2001) 6505-09.-   ¹² S. Rahal, L. Badache: The Eschweiler-Clarke methylation of amino    acids, Journal de la Societe Algerienne de Chimie 4 (1994), 75-85-   ¹³ PA Fowler, A. H. Haines, R. J. K. Taylor, E. J. T.    Chrystal, M. B. Gravestock: Synthesis and biological activity of    acyclic analogues of Nojirimycin, JCS Perkin I (1994) 2229-35.-   ¹⁴ Bohner, M., Ring, T. A., Rapoport, N. and Caldwell, K. D.    “Fibrinogen uptake by PS latex particles coated with various amounts    of a PEO/PPO/PEO triblock copolymer”, J. Biomaterials Sci., Polymer    ed., 13 (2002), 733-746.-   ¹⁵ Andersson, M., Fromell, K., Gullberg, E., Artursson, P., and    Caldwell, K. D. “Characterization of Surface Modified Nanoparticles    for in vivo Biointeraction—A Sedimentation Field-Flow Fractionation    Study”, Analytical Chemistry (2005) 77, 5488-5493.

1. An isolated polypeptide comprising the amino acid sequence according to SEQ ID NO:1, wherein at least one phosphocholine derivative is covalently attached to said polypeptide through a spacer comprising a carbon chain with a length of 1-12 carbon atoms.
 2. An isolated polypeptide consisting of two dimerized polypeptides according to claim 1, wherein the two polypeptides have the same or different amino acid sequences.
 3. The isolated polypeptide according to claim 1, wherein the phosphocholine derivative is attached to a lysine in position 8, 17, 22, or 34 of said polypeptide.
 4. The isolated polypeptide according to claim 1, further having a reporter group covalently attached to said polypeptide.
 5. The isolated polypeptide according to claim 1, having a binding affinity for C-reactive protein of less than 100 nanomolar.
 6. The isolated polypeptide according to claim 2, wherein said polypeptides have the same sequence or different sequences, and said sequences are selected from the amino acid sequences of SEQ ID NOS:3-18.
 7. A method for producing the polypeptide dimer according to claim 2, said method comprising the steps of: synthesizing at least two polypeptides having the sequence according to SEQ ID NO:1; bringing a phosphocholine derivative in contact with an unblocked lysine of one of said polypeptides under conditions suitable for the attachment of the phosphocholine derivative to the lysine; optionally bringing a reporter group in contact with an unblocked lysine of one of said polypeptides under conditions suitable for the covalent attachment of the reporter group to the lysine, and allowing the polypeptides to form a polypeptide dimer.
 8. A method for assaying the concentration of C-reactive protein in a sample, said method comprising the steps of bringing the sample in contact with a polypeptide according to claim 1 and detecting a product obtained by binding of said polypeptide to C-reactive protein in said sample.
 9. The method according to claim 8, wherein the sample is obtained from a mammalian patient.
 10. The method according to claim 9, wherein said polypeptide is bound to a solid support.
 11. A method for assaying the concentration of C-reactive protein in a sample obtained from a mammalian patient according to claim 10, comprising the steps of: bringing the sample in contact with a first polypeptide comprising the amino acid sequence according to SEQ ID NO:1, wherein at least one phosphocholine derivative is attached to said polypeptide, said first polypeptide being bound to a solid support; bringing the sample in contact with a second polypeptide comprising the amino acid sequence according to SEQ ID NO:1 wherein at least one phosphocholine derivative is attached to said polypeptide further having a reporter group covalently attached to said second polypeptide; and detecting the presence or absence of a signal from the reporter group on said second polypeptide.
 12. A composition comprising the isolated polypeptide according to claim
 1. 13. A diagnostic composition comprising a polypeptide according to claim 1, for use in the diagnosis of conditions associated with elevated levels of C-reactive protein, selected from inflammatory conditions, conditions caused by tissue damage, and increased risk of cardiovascular disease.
 14. The isolated polypeptide according to claim 2, wherein the phosphocholine derivative is attached to a lysine in position 8, 17, 22, or 34 of one of the polypeptides.
 15. An isolated polypeptide consisting of two dimerized polypeptides comprising the amino acid sequence according to SEQ ID NO:1, wherein at least one phosphocholine derivative is covalently attached to each polypeptide through a spacer comprising a carbon chain with a length of 1-12 carbon atoms, wherein said polypeptides have the same or different amino acid sequences, and wherein said isolated polypeptide has a reporter group covalently attached thereto.
 16. The isolated polypeptide according to claim 3, further having a reporter group covalently attached to said polypeptide.
 17. The isolated polypeptide according to claim 4, wherein said reporter group is covalently attached to said polypeptide in position 10, 15, 25, or 37 of one of the polypeptides.
 18. A method for assaying the concentration of C-reactive protein in a sample, said method comprising the steps of bringing the sample in contact with a polypeptide according to claim 2 and detecting a product obtained by binding of said polypeptide to C-reactive protein in said sample.
 19. A composition comprising the isolated polypeptide of claim 1 in a pharmaceutically acceptable carrier.
 20. The isolated polypeptide according to claim 1, wherein said polypeptide has a sequence selected from the amino acid sequences of SEQ ID NOS:3-18. 